Device and method for high-intensity homogeneous illumination with minimized light reflection for use in microscopes

ABSTRACT

The invention relates to a device for high-intensity uniform illumination with minimal light reflection for use in reflective-type microscopes. The invention is characterised by a light source ( 1 ) with a uniform emission and the following components, arranged in succession in the emission direction: a lens combination ( 2 ) with a short focal length, the length f 1  being adjusted in such a way that the light source is projected to infinity; a rectangular diaphragm aperture ( 3 ), which is located on the rear focal plane of the lens combination, the Fourier plane of the lens combination being situated on the plane; an additional lens ( 4 ) with a focal length f 2 , through which the rectangular diaphragm aperture ( 3 ) is projected onto the intermediate image plane ( 6 ) of the microscope; and a circular diaphragm ( 5 ), onto which the light source ( 1 ) is projected in sharp focus and which is located on the rear focal plane of the additional lens.

The method described here serves, in microscopes that work with reflection, for suitable formation of the illumination beam at a high light yield, and for adaptation of the illumination aperture to the entry pupil of the microscope lens. Using this invention, it is possible to create new possibilities of use for relatively low-intensity light sources. In particular, the use of LED light sources in microscopes that work confocally, which are considered to be very low-intensity, is made possible by means of this invention. The influence of internal light reflections is significantly reduced by means of suitable diaphragm positioning.

The drawing shows:

FIG. 1) fundamental sketch concerning formation of the light beam for homogeneous image illumination on a defined area

FIG. 2) fundamental sketch concerning projection of the pupils

FIG. 1) shows the principle for homogenization of the illumination of the intermediate image for use in microscopes, with a minimal loss of illumination intensity and minimized formation of reflections. Light that cannot get into the entry pupil of the microscope lens being used is already blocked out by means of the diaphragm combination used, in order to eliminate light reflections. In this case, without any restriction in generality, a LED (1) is to be used as the light source. This should be structured as a Lambert emitter, if possible, and should emit light as homogeneously as possible. A lens combination (2) having as short a focus as possible follows, having the focus f₁ (e.g. f₁=10-15 mm), which is adjusted in such a way that the emitter in the LED is projected into infinity, in other words is at the focus of the lens combination. The sharper the emitter can be projected into infinity, the fewer problems with indefinite light reflections will occur in the following. A rectangular diaphragm aperture (3) is used in the rear focal plane of the lens combination. The aperture must be fully illuminated and is not allowed to be partially shaded by possible additional diaphragms. The Fourier plane of the lens combination (2) is situated in this plane, and for this reason, the illumination is defined, here, by the angle distribution of the emitted light intensity. Because of the Lambert characteristic of the beam profile, very homogeneous illumination of the diaphragm is therefore present at this location. This diaphragm is projected to the intermediate image plane (6) in the microscope, using another lens (4) having the focal width f₂ (e.g. f₂=50 mm). As a result, the intermediate image plane is homogeneously illuminated. A circular diaphragm (5) is used in the rear focal plane of the lens (4). The emitter of the LED (1) is projected sharply on this diaphragm. The magnification of the emitter corresponds to the focal width ratio V=f₂/f₁. At an emitter size of approximately 1 mm (high-power LED), this means illumination of the diaphragm (5) at a diameter of approximately 4-5 mm, at the proposed focal widths. To increase the light yield in the microscope, the diaphragm should be minimally larger, if possible. The magnification V₂=b₂/g₂ that can be achieved with the lens (4) is calculated from the quotient of the desired diagonal of the illuminated intermediate image (6) and the diagonal of the rectangular diaphragm (3). In this connection, b₂ is the distance between intermediate image plane (6) and lens (4), and g₂ is the distance between rectangular diaphragm (3) and lens (4). Therefore, if the intermediate image is supposed to have a diagonal of 22.6 mm at a diaphragm diagonal of 11.3 mm, the magnification turns out to be V₂=2, in other words b₂=2·g₂ at g₂=1.5·f₂ and b₂=3 ·f₂. The distance between perforated diaphragm (5) and intermediate image (6) is calculated at d=b₂−f₂=2·f₂.

FIG. 2) shows a typical beam path in a reflection-light microscope or a confocal microscope, with illumination through the microscope lens. In this connection, the perforated diaphragm (5), which corresponds to the perforated diaphragm (5) from FIG. 1), is projected onto the entry pupil of the microscope lens (9), by means of the field lens (8). The image of the emitter is therefore situated on the entry pupil of the lens, shows itself behind the lens as an angle distribution, and only becomes clearly visible behind the focal plane of the lens. The illumination of the sample approximately corresponds to the illumination in the intermediate image plane (6), where the confocal filter disk is typically situated. In the detection branch, the light reflected back by the sample is first projected onto the intermediate image plane (6). The field lens (8), which is situated close to the intermediate image plane, having the focal width f₃, focuses the pupil of the microscope lens onto the pupil of the projection lens (10), by way of the beam splitter. The projection lens (10) projects the intermediate image (6) onto the detector matrix (11), typically a CCD camera. The optical path that lies between intermediate image plane and diaphragm (5) and projection lens (10), respectively, should have an identical length, in order to achieve maximal illumination.

At a tube length of 160 mm, for example, a magnification of the diaphragm (5) of maximally 1.6 is obtained, to continue the above example. This means that a diaphragm diameter (5) of 5 mm in the illumination branch is projected onto a pupil diameter of 8 mm, and focused onto a spot diameter again in the detection branch.

At f¹=b⁻¹+g⁻¹, a focal width f₃ of 61.5 mm is obtained for the case that the field lens is situated approximately in the intermediate image plane. 

1: Device for high-intensity homogeneous illumination, with minimized light reflections, for use in microscopes that work reflectively, comprising a light source (1) that emits homogeneously, having a lens combination (2) with a short focal width disposed subsequently in the emission direction, with a focal width f₁, which is adjusted in such a way that the light source (1) is projected into infinity, and in the rear focal plane of which a rectangular diaphragm aperture (3) is used, whereby the Fourier plane of the lens combination (2) is situated in this plane; by an additional lens (4) having the focal width f₂, by means of which the rectangular diaphragm (3) is projected onto the intermediate image plane (6) of the microscope (9), and in the rear focal plane of which a circular diaphragm (5) is disposed, onto which the light source (1) is sharply projected. 2: Device according to claim 1, wherein the light source is the emitter of an LED. 3: Device according to claim 1, wherein the light source (1) is structured as a Lambert emitter. 4: Device according to claim 1, wherein a field lens (8) is disposed between the circular diaphragm (5) and the lens of the microscope (9), and a beam splitter (7) is provided between the circular diaphragm (5) and the intermediate image plane (6), by way of which splitter the pupil of the microscope lens is projected onto the pupil of a projection lens (10) having the focal width f₃, which lens projects the intermediate image (6) onto a detector (11), which can be a CCD camera, for example. 5: Device according to claim 4, wherein the optical path between the intermediate image plane (6) and the diaphragm (5) is identical with the optical path between the intermediate image plane (6) and the projection lens (10). 